Figure 6. Representative SPR sensorgrams for Nef PROTACs.
The thalidomide-binding domain of Cereblon (CRBN-TBD) and full-length Nef (NL4-3 variant) were expressed in E. coli and purified to homogeneity. A) Each protein was immobilized on one channel of a carboxymethyl dextran biosensor chip, and the two Nef PROTAC analogs FC-12988 and FC-13182 (structures at top; Nef-binding moiety in red, CRBN-binding ligands thalidomide and lenalidomide are shown in green and blue, respectively) were injected over the range of concentrations shown in the upper left sensorgram. Protein-ligand interaction was followed for 90 s, followed by a 180 s dissociation phase. The resulting data were fit to a 1:1 Langmuir binding model, and KD values were calculated from the resulting association and dissociation rate constants (KD values are summarized in Table S2). Each concentration was tested in triplicate, and individual traces are shown with the data shown in color and the fitted curves superimposed in black. RU, SPR response units. Arrows indicate the point of injection and the switch to wash buffer for dissociation. B) Comparison of SPR profiles for interaction of active PROTAC analog FC-14369 with NefNL4-3 vs. inactive analog FC-13906. The inactive analog dissociates rapidly from Nef while the active analog remains bound. Additional examples are provided in Figure S5.